Treatment of mammalian reaction of IgE interactions

ABSTRACT

A method is disclosed for blocking or reducing physiological reaction in a mammal to the interaction of IgE antibodies present in said mammal upon contact with the corresponding antigen, by the administration to said mammal of a therapeutically effective amount of a neurotoxin (CnT) derived from Clostridia sp.

FIELD OF THE INVENTION

A method is disclosed for blocking or reducing physiological reaction ina mammal to the interaction of IgE antibodies present in said mammalupon contact with the corresponding antigen, by the administration tosaid mammal of a therapeutically effective amount of a neurotoxin (CnT)derived from Clostridia sp.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to withinparentheses or by footnote. The contents of these publications arehereby incorporated by reference in their entirety.

INTRODUCTION

The physiological reaction in a mammal to the interaction of IgEantibodies present in said mammal upon contact with the correspondingantigen is generally referred to as allergy. It is believed that theallergic response evolved to combat infections with parasites. Asparasitic infections are rare in industrialized countries this mechanismbecomes pathologic in certain susceptible (atopic) individuals.Characteristic of allergies is that the antigen, also called theallergen, is normally innocuous, and the body's reaction isinappropriate.

Allergic reactions can involve all body tissues but are most prominentin the boundaries with the external environment: the airway, eye,gastrointestinal tract and skin. The conditions are known as allergicrhinitis in the nose (hay fever), allergic asthma in the lungs, foodallergies in the GI tract and allergic hypersensitivity (hives) ordermatitis in the skin. Anaphylaxis refers to the systemic reaction thatoccurs when an allergen is introduced directly into the circulation,such as occurs with injection of drugs and insect stings. A massiveearly phase reaction occurs throughout the body. Symptoms includediffuse airway swelling and hypotension which can be life threatening.

Each year more than 50 million Americans suffer from allergic diseases,costing the health care system $18 billion annually (American Academy ofAllergy, Asthma and Immunology (AAAAI). The Allergy Report: ScienceBased Findings on the Diagnosis & Treatment of Allergic Disorders,1996-2001)

Alarmingly, the prevalence and severity of allergic disorders hasrapidly increased in the United States over the past twenty years, sothere is a need for new therapies for these conditions.

In 1998, an estimated 15 million Americans, or 6.4 percent of thepopulation, had asthma, with 5 million being children. Children accountfor 4.8 million of Americans with asthma. Each year, 1.8 millionemergency room visits are for asthma, nearly 500,000 Americans arehospitalized and more than 5,000 die from the disease. Although asthmadeaths are infrequent, they have increased significantly during the lasttwo decades. From 1975-1979, the death rate was 8.2 per 100,000 people.That rate jumped in 1993-1995 to 17.9 per 100,000. Asthma cost the U.S.economy an estimated $10.7 billion in 1994, including a direct healthcare cost of $6.1 billion and indirect costs, such as lost workdays, of$4.6 billion.

Approximately 16.7 million office visits to health care providers eachyear are attributed to allergic rhinitis alone (CDC. Fast Stats A-Z,Vital and Health Statistics, Series 10, no. 13. 1999

A related condition, chronic sinusitis, is the most commonly reportedchronic disease, affecting 12.6 percent of people (approximately 38million) in the United States in 1996. Another related condition, serousotitis media, is the most common condition in children requiring anoffice visit to a health care provider.

Atopic dermatitis is one of the most common skin diseases, particularlyin infants and children. The estimated prevalence in the United Statesis 9 percent Rudikoff D and Lebwohl M. “Atopic dermatitis.” Lancet351(9117): 1715-21. 1998.

Experts estimate that food allergy occurs in 8 percent of children 6years of age or under, and in 1 to 2 percent of adults Sampson H A. InAllergy, Principles and Practice, 5^(th) Ed., E. Middleton et al, ed.Mosby, St. Louis, p. 1162. 1998.

Pathophysiology of Allergy

Allergy is an ailment that affects millions of individuals worldwide.Attempts to desensitize an individual against a material that causes anallergic response (hereafter designated as an “allergen”) by injectionof measured dosages of the allergen heretofore has failed to achievecomplete relief of allergy symptoms reproducibly in all allergicindividuals. An allergic response is a term of art and has awell-defined meaning. Within the context of the present invention, anallergic or reagenic response includes, in particular, at least one ofthe features of 1.) production of an abnormally high level of IgE in anindividual's serum, 2.) immunologic interaction between an allergen, anindividual's IgE and leukocytes resulting in release of histamines, 3.)production of hives, rashes, wheal and flare and similar dermatologicalmanifestation of hypersensitivity, and 4.) anaphylaxis.

An allergic response is a state of hypersensitivity in which anexaggerated immune response is induced by the exposure to a particularantigen or allergen. Hypersensitivity reactions can be classified asimmediate or delayed. Immediate or type I hypersensitivity (oranaphylactic response) is an allergic reaction which develops veryquickly, i.e., within seconds or minutes of exposure of the patient tothe causative allergen, and it is mediated by IgE antibodies produced byB lymphocytes. In non-allergic patients, there is no IgE antibody ofclinical relevance. However, in a person suffering with allergicdiseases, IgE antibody mediates immediate hypersensitivity bysensitizing mast cells which are abundant in the skin, membranes of theeye, nose and mouth, and in the respiratory tract and intestines.

IgE secreted from activated B cells can attach to Fc receptors locatedon the surface of mast cells and basophil granulocytes, which containnumerous cytoplasmic granules packed with chemical mediators e.g.histamine (J. Klein, “Immunology”, Blackwell Sci. Pub., London, 1990; E.Benjamini & S. Leskowitz, “Immunology”, Wiley-Liss, N.Y. 1991). Thisreceptor binds circulating IgE with very high affinity and retains it atthe mast cell surface for extended periods of time. Activation isaccomplished through the binding of an allergen simultaneously to morethan one polyvalent molecule of Fc receptor-bound IgE. This “crosslinking” of at least two surface-bound IgE molecules brings Fc receptorproteins into close association with one another in the plane of themast cell plasma membrane. When the bound IgE is contacted by theappropriate allergen, the mast cell becomes activated Kinases associatedwith these receptors become activated as a result of this proximity.They initiate the second messenger cascade, which results in the fusionof the granules with the cell surface membrane, leading to theexocytosis of the granule contents, such as histamine, cytokines, andleukotrienes into the surrounding tissue, and the concomitant inductionof allergic symptoms. It is the activity of these substances which isresponsible for the clinical symptoms typical of immediatehypersensitivity. These include contraction of smooth muscle in theairways or the intestine, the dilation of small blood vessels and theincrease in their permeability to water and plasma proteins, thesecretion of thick sticky mucus, and in the skin, swelling and thestimulation of nerve endings that results in itching.

Delayed type hypersensitivity (DTH) reactions are mediated by T-cellsand macrophages and become evident only after 1 to 2 days and persistfrom several days to a few weeks. DTH is also referred to ascell-mediated hypersensitivity (i.e., T-cell mediated) and is part of alarger group of reactions called cell-mediated immunity.

Anaphylaxis, or anaphylactic shock, is an acute systemic (whole body)type of allergic reaction, It occurs when a person has become sensitizedto a certain substance or allergen (that is, the immune system has beenabnormally triggered to recognize that allergen as a threat to thebody). On the second or subsequent exposure to the substance, anallergic reaction occurs. This reaction is sudden, severe, and involvesthe whole body. Anaphylaxis is life-threatening and can occur at anytime.

Therapeutically, many agents are used to try to prevent the release ofmediators from mast cells and basophils and/or to treat the downstreamevents by blocking or ameliorating the effects of the mediators ontarget tissues. Therapeutic agents commonly employed fall under thefollowing main groups.

Antihistamines block and mop up the released histamine, i.e. the majormediator of the allergic response.

Alpha-1 beta-2 agonists, e.g., Epinephrine, Salbutamol overcomeindirectly the downstream effects on vasculature and smooth muscle.

Chromoglycate is useful for primary prevention of mast cells/basophildegranulation. This prophylactic must be taken continuously. It does notprevent the cross-linking of IgE but it somehow interferes withsubsequent events. Theophylline and other phosphodiesterase inhibitorsagain influence downstream biochemical events particularly associatedwith cyclic nucleotides. Steroids have multiple sites of activitiesagainst the allergic response. They are either administered locallyand/or systemically.

None of the above treatments are ideal for the modulation of allergicresponses because each has specific problems such as side effectsincluding drowsiness, they also lack specificity in the immune systemleading to global immuno-suppression. Also many of these therapeuticagents need to be administered continuously. Therefore, new improvedtreatments are constantly being sought to control the allergic responseprophylactically and/or therapeutically without the above-mentionedlimitations.

Individuals who wish to become desensitized against an allergen oftenmust submit himself/herself to injections of measured doses of theallergen, first administered at weekly or biweekly intervals, thengradually decreases to bimonthly or monthly intervals throughout theyear. Such injections generally commence with a small dose of theallergen and then gradually increasing the dosage until amaximally-tolerated maintenance dose is achieved. The individual is thenkept on a maintenance dose of the allergen for long periods of time oruntil the individual no longer exhibits an allergic reaction to theallergen.

Other treatment regimes have been devised to reduce or eliminate anallergic response.

Allergen injection therapy (allergen immunotherapy also known assubcutaneous immunotherapy (SCIT) is known to reduce the severity ofallergic rhinitis. This treatment is theorized to involve the productionof a different form of antibody, a protective antibody which is termed a“blocking antibody” (Cooke, R A et al., Serologic Evidence of Immunitywith Coexisting Sensitization in a Type of Human Allergy, Exp. Med.1935; 62:733). Chemical agents have been developed which inhibit theinteractions between the IgE and its receptor (Cheng et al., U.S. Pat.No. 5,965,605 and Ra et al., U.S. Pat. No. 6,090,034). IgE antagonistshave also been used to treat allergic disease (Presta et al., U.S. Pat.No. 5,965,709) and compounds that exhibit immunosuppressive activity andinhibits the release of histamine (Bycroft et al., U.S. Pat. No.5,969,158). St. Remy et al., U.S. Pat. No. 4,740,371, discloses animmune complex of an allergen for treating allergies involving acombination of the specific allergen and the corresponding antibody tothat allergen. The injection of the complex into a patient is said toreduce a patients allergic reaction to that specific allergen. Othershave suggested that certain human proteins can neutralize IgE byblocking it from interacting with the mast cells, but this has not beenestablished clearly as a clinically effective therapy (Stanworth, etal., Allergy Treatment with a Peptide Vaccine, Lancet 1990;336:1279-81). Patterson et al., U.S. Pat. No. 5,314,690 disclosed amethod and preparations for reducing IgE antibodies to allergens inallergic subjects wherein substance P (a neuropeptide) and an allergenor fragments of allergens or haptens acting as allergens areadministered together to the allergic subjects through a non-oral route.

Cholera Toxin and B Subunit as Adjuvants Cholera toxin (CT) and theclosely related heat-labile toxin (LT) from Escherichia coli are knownas exceptionally potent immunoadjuvants when co-administered withantigens by various mucosal routes (Elson et al., J. Immunol. 1984;133:2892-2897; Hohngren et al., Vaccine 1973; 1 1: 1 179-1184; Lycke etal, Eur. J. Immunol. 1992; 22:2277-228 1. Both CT and LT are recognizedas “AB” toxins in that they are composed of two distinct structural andfunctional components: a single toxic-active A subunit component and anon-toxic cell-binding B subunit. The latter is a homopentamer componentwith strong binding affinity for GMI ganglioside receptors (Holingren etal., Nature 19 8 1; 292:413-417). Such receptors are known to be presenton most mammalian cells, e.g., on skin and other epithelial cells, onall known antigen-presenting cells including dendritic cells (DC) andLangerhan's cells (LC), as well as on B and T lymphocytes.

Recently, Gleim et al., U.S. Pat. No. 5,980,898 disclosed a system fortranscutaneous immunization that induces an immune response (e.g.,humoral and/or cellular effectors) in an animal or human. The systemprovides a simple application to intact skin of both rodents and humansof a formulation comprised of antigen and an adjuvant that was wholecholera toxin.

Common to allergies is the involvement of the IgE class of antibody.Individuals are not born with allergies; rather they acquire them byexposure to allergens. The steps of the IgE allergic reaction aresensitization upon first exposure to the allergen, and then the allergicresponse to subsequent exposures. The allergic response consists of animmediate and delayed response referred to as the early and late phaseresponses respectively. In atopic individuals, those prone to allergies,the initial exposure to an antigen results in the production of IgEantibodies that specifically recognize that allergen. This process iscalled sensitization.

The early phase response (ERP) is the immediate reaction that occurswithin minutes of exposure to an allergen. IgE are bound to the surfaceof a neuroimmune cell called the mast cell (in the circulation thesecells are called basophils). Sufficient numbers of bound IgE antibodiesthat react with an allergen causes the mast cell to release its contentof secretory vesicles, a process known as degranulation. The secretoryvesicles contain histamine and other stored substances such as nervegrowth factor (NGF). In addition the mast cell and T cells immediatelybegin manufacturing leukotrienes, cytokines, enzymes and substances thatactivate blood platelets and attract secondary cells such aseosinophils. Symptoms vary depending on the site, but common reactionsare smooth muscle contraction, mucus secretion, vascular permeability,and sensory nerve stimulation.

The late phase response (LPR) develops over hours to days of exposure aseosinophils and other cells are attracted to the area. Eosinophilsproduce major basic protein, eosinophil cationic protein, leukotrienesand nerve growth factor. TH2 lymphocytes release cytokines that promotefurther IgE production and eosinophil chemo attraction, and increasednumbers of mast cells.

Nerve Involvement in Allergy

The sensory nerve stimulation causes reflexes that are designed to aidin defending the tissue. These reflexes are often a larger problem thenthe local allergic response. Reflexes can range from large gross motoractions to regional afferent and efferent arcs or even local axon-axonalreflexes involving a single neuron.

Some reflexes recruit major motor actions that are well recognized. Inthe nose, sneezing is a reflex attempt to expel unwanted material andcoughing is the equivalent response in the lungs.

Regional reflex arcs involve the sensing of the stimulus by the sensoryneuron, the transfer of the message to the ganglia and the centralnervous system and an efferent response via autonomic neurons. Reflexexcitation by the autonomic nervous system directly causes mast cell todegranulate, thereby spreading the reaction. In addition these reflexescontrol a variety of other functions. In the nose these reflexes causeincreased mucus production, increased cilia movement, nasal congestionand sneezing. In the lungs reflexes cause bronchospasm, increasedmucosal congestion, production of airway secretions and coughing. In theGI tract reflexes cause dysmotility, mucosal congestion and secretions.In the skin the reflexes cause swelling and itching.

Finally there are local axon-axonal reflexes in sensory nociceptivenerve fibers. Allergic stimulation of a single neuron causes release ofmediators from other axons of the same neuron. (Barnes P et al. 1991Neuropeptides in the respiratory tract. Am Rev Resp Dis 144:1187-1198,1391-1399)

In chronic allergic stimulation the mast cells and eosinophils releasesnerve growth factor which causes growth of the nerves in the region.Thereby allowing for increased neural responses and hyper reactivity.This hyper reactivity is not limited to allergic reactions but extendsto non-allergic conditions such as respiratory tract infectionsincluding viral and bacterial infections. Specifically viral rhinitis,viral and bacterial sinusitis, suppurative otitis media, bronchitis andpneumonia. Therefore individuals become more susceptible to theseconditions and have more frequent and severe infections.

Furthermore the repeated allergic reactions cause changes in thequalitative response of the neural reflexes such that they areinappropriately activated. This negatively effects non-allergicconditions, such as bronchospasm mucus production and coughing innon-allergic lung conditions such as bronchitis and emphysema.

Allergic Reactions Differ from Inflammatory Reactions

Allergic reactions differ from inflammation. Allergies represent thebody's inappropriate response to what is in essence a harmless antigen.It is believed that the IgE allergic reactions evolved to combatparasitic infections which are now rare in industrialized societies. Incontrast, inflammation is the body's response to actual tissue damage orinfection. Inflammation is clinically distinguished by the classicalsymptoms of calor, rubor and dolor heat, redness and pain. Inflammationis a process that triggers a cascade of mediators with wide effects,both locally and systemically. Local reactions include pain,vasodilation and migration of macrophages and neutrophils. Systemicreactions include fever. Certain conditions, such as rhinitis or asthma,can be triggered by allergies, infections, irritating chemicals or maybe entirely neurogenic, the result of nerve activity without priorinflammatory or allergic stimulation.

Specific Allergic Conditions

Rhinitis

Allergic Rhinitis

The inner lining of the nose is a mucosa that contains serous and mucusglands and large numbers of mast cells. This mucosa extends to theopenings leading to the sinuses as well as the Eustachian tube where itis continuous with the mucosa of the sinuses and middle ear,respectively. The EPR of the nasal mucosa causes mast celldegranulation. The release of histamine, heparin and neuropeptidesprovokes vasodilation and acute swelling of the mucosa and has someminor direct stimulatory effect on the mucus secreting glands. Reflexexcitation causes reflex sneezing, congestion and neural stimulation ofthe seromucinous glands and further congestion. LPR attracts eosinophilsand prolongs the reaction.

Allergic reactions can also be chronic, a condition known as perennialrhinitis. In these cases airborne allergens are constantly in theenvironment. Chronic low-level allergic reactions cause a thickening ofthe nasal mucosa due to edema and hypertrophy of glandular elements. Asa result the primary symptoms of perennial rhinitis are nasalcongestion, and postnasal drip. After each allergic reaction the mucosaswells; repeated allergic reactions enlarges the mucosa permanently andmay form polyps. Thickening of the mucosa can cause obstruction of thesmall openings that allow drainage from the sinus. Obstruction of theseopenings allows nasal secretions to collect and become infected, therebycausing sinusitis.

Topical treatment of allergic rhinitis includes steroid sprays andchromalyn sodium (Nasocrom®) a chemical that blocks mast celldegranulation, and/or nasal decongestants (Neosynephrine). Systemictreatment includes oral anti-histamines and non-sedating antihistamines(Allegra®, Zyrtec®, Claritin®). Long-term therapy requires immunedesensitization to the allergen by progressive intradermal injections ofthe allergen over months to years. rhinitis can lead

Allergic rhinitis can lead to pulmonary disease, including but notlimited to asthma and emphysema, suitably asthma caused byhyperreactivity and/or the symptoms of bronchoconstriction, mucosaledema, increased secretions and cough. These can be treated by theapplication of CnT to the nasal cavity of patients with allergicrhinitis. It is well known in the art that allergic conditions of thenose can cause reflex changes in the lung that mimic or exacerbateasthma. (McCusker C et al 2002, Site-specific sensitization in a murinemodel of allergic rhinitis: Role of the upper airway in lower airwaydisease, J Allergy Clin Immunol, 110:891-898)

Perennial Allergic Rhinitis

Perennial rhinitis is a chronic condition and certain symptoms, such asnasal congestion, are more prominent then others such as sneezing.Therefore therapy for this condition needs to be over more prolongedperiod, if not indefinitely. Although the same treatments as disclosedfor seasonal allergic rhinitis can be used repeatedly, certain methodsof treatment are disclosed here that are more efficient and convenientfor longer term therapy.

Parasympathetic neurons do not extend from the central nervous system totheir target organs in the nose. Instead parasympathetic preganglionicneurons have their cell bodies in the brain stem and have an axon thatextends partway and synapses on the cell body of a second neuron calledthe postganglionic neuron. The axons of the postganglionic neuronsextend into nose and sinuses and modulate many of the biologicalresponses seen during allergic reactions. The cell body of the postganglionic parasympathetic neuron also receives collateral afferentsynapses from sensory neurons that are stimulated during allergicreactions. Therefore much of the neuronal circuit involved in theallergic reaction converges onto the cell body of the postganglionicneuron.

All of the postganglionic neuron cell bodies are concentrated in smallstructure called the sphenopalatine ganglion. Moreover all afferentsynaptic connections on these neurons is cholinergic, theneurotransmitter most sensitive to BoNT. Therefore application of CnT tothe sphenopalatine ganglion is the most efficient way of blocking theallergic neural reaction.

The sphenopalatine ganglion is accessible to needle injection via thesphenopalatine canal. This canal passes though the hard palate and isaccessible about one centimeter medial to the second molar. Injectionsof anesthetic and vasoconstrictor agents are commonly performed byclinicians skilled in the art of nasal surgery.

Infectious Rhinitis

Although they share some of the same symptoms, allergic rhinitis differsfrom infectious rhinitis. The most common cause of infectious rhinitisis a viral infection, known as the common cold. Bacterial and fungalinfections of the nose occur also but these are comparatively rare asprimary events. During viral infections the epithelial cells releasesecretions that trigger an inflammatory response. The classic signs ofcaldor, rubor and dolor; heat, redness, and pain characterizeinflammation. White blood cells such as macrophages and neutrophilsmigrate from small blood vessels in the area to the infected mucosa.These cells in turn release a variety of chemical mediators thatincrease the inflammation. Infectious rhinitis is characterized by apurulent or pus like postnasal drip, and nasal congestion. In many viralinfections there are systemic symptoms such as fever. Treatment of viralinfections is symptomatic with topical and systemic decongestants, andantipyretic and anti-inflammatory drugs.

Vasomotor and Other Causes of Rhinitis

Vasomotor rhinitis is strictly a neural hyper stimulation of the seroussecretory glands of the nose. This condition is characterized by awatery nasal discharge with a very small content of mucus. Vasomotorrhinitis results directly from neural stimulation of temperature changesor while eating, and associated other symptoms such as congestion, andsneezing are not prominent. U.S. Pat. No. 5,766,605 discloses a methodof blocking the symptom of rhinorrhea in vasomotor rhinitis usingclostridia neurotoxins.

Serous Otitis Media

Direct reaction of the middle ear mucosa to allergens, or indirecteffects by blockage of the Eustachian tube, leads to serous otitismedia. In this condition the middle ear fills with fluid and hearing isimpaired. As this condition most often affects young children they areat risk of learning disabilities due to the decreased hearing acuity.

Sinusitis, a Complication of Rhinitis

Sinusitis is an infection of the sinuses. The sinuses are air filledcavities surrounding and opening into the nasal cavity via smallopenings called ostia. The maxillary sinuses compose the mass of thecheeks and the ostia are between the inferior and middle turbinate. Thefrontal sinuses are above the brow underneath the skin of the forehead,they open into the nasal cavity via a duct that enters beneath themiddle turbinate. The sphenoid sinuses are in the nasopharynx and haveostia on their anterior surface. The ethmoid sinuses are located betweenthe eyes and are composed of numerous small air cells. The anterior andposterior cells drain independently. The anterior ethmoid region isespecially important due to the confluence of drainage ostia. The areais called the infundibulum, and a small amount of obstruction can causewidespread sinus infections.

Sinus infections are either acute or chronic. In acute sinusitis abacterial infection follows a viral infection of the upper respiratorytract. However, sinusitis can also occur from obstruction of thedraining ostia of the sinuses by inflammation or mass lesion. Topicalnasal decongestants and systemic antibiotics are used to treat acutesinusitis. Severe or complicated infections may require surgicaldrainage of the involved sinus. Chronic sinusitis is a low-grade ongoingor recurrent infection of the sinuses. In these cases the mucosa of thesinuses has become thickened and enlarged due to edema and increasedsecretory glandular elements. In these cases small abscess may persistand relatively mild inflammatory stimuli may cause obstruction of thenarrowed sinus ostia. Treatment of chronic sinusitis includes topicalsteroid sprays, systemic anti histamines and decongestants, and/orsurgical removal of diseased mucosa with enlargement of the ostia.

Asthma

Asthma is a general term given to a similar constellation of symptoms:bronchospasm, mucosal swelling and increased secretions. Allergicstimulation, infections, irritating chemicals, cold air or exercise cantrigger asthma.

Allergic asthma is a specific subset of asthma that is initiated byallergens and involves the IgE mediated reaction. Local and organreactions include bronchial smooth muscle constriction, mucosal swellingand increased secretions.

Other Allergic Conditions

Other allergic conditions include food allergies, and allergicdermatitis.

Clostridia Neurotoxins (CnT)

Clostridium (C.) botulinum and the closely related species C. butyricumand beratti produce an extremely powerful neurotoxin that causes theparalytic condition known as botulism. The botulinum neurotoxin (BoNT)protein consists of a light and heavy chain that together weighapproximately 150 kD. In botulism the primary target is the synapse ofthe motor neuron with the muscle fiber. Here BoNT is taken up by themembrane of the motor neuron and is internalized. The effect of BoNT isto inhibit the release of neurotransmitters and neuropeptides byneurons. In clinical use each serotype appears to differ in its potencyin blocking different classes of neurons.

BoNT works by a two-stage mechanism, uptake and molecular action.Peripheral nerve terminals take up BoNT. After translocation across thecell membrane BoNT interferes with the molecular mechanism ofneurosecretion. Specifically, BoNT cleaves the proteins involved insynaptic vesicle docking and release called the SNARE complex. Theresult is to block neural signals.

C. tetani produce tetanus neurotoxin (TeNT). TeNT is similar to theBoNTs in that it interferes with vesicle release by cleaving VAMP, oneof the SNARE family of proteins. However the in vivo biological activityof TeNT is usually quite different from BoNT. The systemic disordertetanus results from TeNT produced at a wound site and disseminatedthroughout the body via the blood stream. The TeNT is taken up byperipheral motor neurons and transported to the central nervous system.The TeNT then preferentially blocks inhibitory neurons connecting to themotor neuron, thereby allowing unopposed excitatory input. However, athigher doses TeNT and when introduced directly into the neuron alsoblocks all neurons in the same manner as BoNT. In this application it isassumed that the when BoNT is discussed it includes the TeNT when usedat higher blocking doses.

At present seven immunologically distinct serotypes of the BoNT areknown, named A, B, C, D, E, F and G. The type C serotype is now known tobe divided into three different toxins with distinct biological effects.Only C1 is a neurotoxin, whereas C2 and C3 are not. C2 is distinctivefor blocking actin formation, which can prevent mast cell degranulation.Although all BoNT serotypes interfere with proteins that cause therelease of synaptic vesicles from cells they each interfere withdifferent proteins, or different parts of the same protein:

BoNT A & E cleave SNAP-25 (synapse associated protein)

BoNT C cleaves SNAP-25 and syntaxin

BoNT B, D, F & G (and TeNT) cleave VAMP (vesicle associated membraneprotein)

Most if not all cell types use the vesicle system for secretion,although the molecules within these vesicles differ for each type ofcell. If experimentally introduced into any cell BoNT appears capable ofblocking its vesicle release. However, in nature BoNT appears to beinternalized into neurons, particularly the efferent neurons. Thevesicles within neurons contain classical neurotransmitters(acetylcholine, epinephrine, nor epinephrine, dopamine, serotonin,glutamate, GABA and others) and/or neuropeptides (substance P,neurokinin A, calcitonin gene related peptide (CGRP), neuropeptide Y,interleukins, growth factors and others). Although the highest affinityof BoNT is for cholinergic neurons, in various preparations BoNT hasbeen shown to block secretion of all these molecules.

The Clinical Effects of Botulinum Toxin on Different Classes of Neurons

Voluntary Motor Nerves

The first and still primary use of BoNT is to block motor nervecommunication with muscle fibers. BoNT is injected within the targetmuscle. The BoNT is then internalized into motor neurons where itdecreases or stops the release of the neurotransmitter acetylcholine(AChE), thereby causing paresis or paralysis of the muscle. Scottintroduced the concept of localized muscular injections of BoNT in thespecific condition of strabismus (squint, crossed eyes). Later BoNT wasfound to be particularly useful for movement disorders such as tics,spasms, contractures, cramps and tremors. More recently, the injectionof BoNT into facial muscles has been found to ameliorate skin wrinklingand lines related to aging. Another recent application of BoNTinjections is to decrease the pain accompanying muscle tension inconditions such as headache and temporo-mandibular joint syndrome.

Autonomic Motor Neurons

Effector neurons of the autonomic system innervate and control thecontraction of smooth muscles using AChE as the neurotransmitter.Injections of BoNT have been used to decrease tone in the smooth musclesof the lower esophageal sphincter, esophagus, stomach wall, and pyloricsphincter, sphincter of Odi, anal sphincter, and urinary bladder.

Autonomic Secretory Neurons

Effector neurons of the autonomic system control or modulate thesecretion of various water and mucoid fluids throughout the body. BoNTinjections have been used to decrease sweating, salivary gland flow,gastric secretions including acid production, nasal and otherrespiratory secretions, and tearing.

Sensory Neurons

Sensory neurons release a wide variety of neuropeptides, cytokines,growth factors and other substances that effect parenchymal cells, bloodvessels and immune cells. Notable is that these substances can activatemast cells directly thereby extending the allergic reaction. Except fornitrous oxide these substances are released via the SNARE mechanism andcan be blocked by CnT.

SUMMARY OF THE INVENTION

As used herein ‘local administration’ includes but is not limited toinjection by needle and in dwelling catheter (including pressure jetinjectors), topical administration in lyophilized powder, liquidsolutions, creams, ointments, aerosolized or introduced by liposomal(niosomes) vectors, or as nucleic acid introduced by viral or othervectors. The CnT may also be embedded in biopolymers or delivered byimplanted pumps to release the CnT into solution over prolonged periods.

By “therapeutically effective amount” it is meant of purposes of thisinvention that the CnT is administered in a non-toxic amount sufficientto cause reduction in the occurrence or magnitude of the symptoms beingtargeted.

By “unit” it is meant the biological equivalent of the current unitmeasure used for botulinum toxin A marketed as Botox. At present BoNT ismeasured by biological assay; a unit of BoNT is the amount that causesdeath to 50% of mice when injected intraperitoneally. BoNT-A is marketedas Botox by Allergan Corp, Irvine Calif., and as Dysport by Ipsen Ltd,Berks United Kingdom. Although the biological assay is done the same waythe in vivo effect of Botox and Dysport vary. BoNT-B is marketed asMyobloc by Elan Pharmaceuticals, Dublin, Ireland. TeNT is notcommercially available but other assays have compared the potency of theblocking effect of TeNT to BoNT. All serotypes of BoNT as well as TeNTare commercially available from List Biological laboratories. Atherapeutically effective amount of BoNT will vary depending on theorgan to be treated, how much of the organ will be treated, the methodof application and the exact preparation of BoNT used. A therapeuticallyeffective amount will vary from a fraction of a unit to hundreds ofunits as it currently does with intramuscular injections. The exactdosage will not require undo experimentation by those skilled in theart.

Where solutions or suspensions of BoNT or CnT are referred to, unlessindicated to the contrary, this means the designated number of units in1 ml of Normal saline.

By “CnT” it is meant that any biological substance having essentiallythe same biological effect within cells as the wild types of clostridianeurotoxins. Specifically, to block or decrease the activity of theSNARE family of proteins involved in secretion of allergy relatedneurohumors. Numerous substitutions for the major parts of the CnT havebeen disclosed and these are all included in this specification. Thiswould include fragments, altered forms, and recombinant forms of CnT.Also included are chimeras, hybrids and conjugates. Also included arethe use of DNA and RNA sequences that are directly applied andtranslated in the allergic sites. Also included are “vectors”, variouscompositions that deliver a botulinum or tetanus toxin light chain orits equivalent such as Protease A across cell membranes. These vectorsinclude but are not limited to viruses, liposomes, noisomes, and proteintransduction domains (U.S. Provisional Application 60/449,107).

Allergic “neurohumors” are neurotransmitters, neuropeptides andcytokines that participate in allergic reactions and whose secretion oraction can be blocked by CnT. They include acetylcholine, noradrenaline,neuropeptide Y, substance P, calcitonin gene reactive protein (CGRP),histamine, nerve growth factor, and interleukins. The invention isdirected to a method of blocking or reducing physiological reaction in amammal, suitably but not limited to H. sapiens, to the interaction ofIgE antibodies present in said mammal upon contact with thecorresponding antigen. This blocking is achieved by the administrationto said mammal of a therapeutically effective amount of a neurotoxin(CnT) derived from Clostridia sp. Suitably, the CnT is derived from aspecies of Clostridia selected from the group consisting of C.botulinum, C. butyricum, C. beratti, C. tetani. The neurotoxins (BoNT),derived from C. botulinum, are derived from serotypes A, B, C1, D, E, Fand G, while neurotoxin (TeNT), is derived from C. tetani

BoNT/A is marketed as Botox® by Allergan Inc and as Dysport® by IpsenLtd as a lyophilized powder that is reconstituted with preservative freenormal saline prior to use. BoNT/B is marketed as Myobloc® by ElanPharmaceuticals in normal saline solution. The light chains andholotoxins for each BoNT serotypes and TeNT can be obtained from ListBiological Labs and/or Metabiologics Inc.

CnT compositions of the present inventions are prepared in a variety offorms depending on whether the composition is injected or implanted,topically applied to respiratory mucosa of the nasal cavity or lungs,gastrointestinal mucosa, or to skin.

For all injectable CnT compositions fluid dosage forms are preparedutilizing the compound and a pyrogen-free sterile vehicle. The compound,depending on the vehicle and concentration used, can be either dissolvedor suspended in the vehicle. In preparing solutions the compound can bedissolved in the vehicle, the solution being made isotonic if necessaryby addition of sodium chloride and sterilized by filtration through asterile filter using aseptic techniques before filling into suitablesterile vials or ampoules and sealing. Alternatively, if solutionstability is adequate, the solution in its sealed containers may besterilized by autoclaving. Advantageously additives such as buffering,solubilizing, stabilizing, preservative or bactericidal, suspending oremulsifying agents and/or local anaesthetic agents may be dissolved inthe vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicleprior to use, may be prepared by filling pre-sterilized drug substanceand other ingredients into a sterile container using aseptic techniquein a sterile area. Alternatively the drug and other ingredients may bedissolved into suitable containers using aseptic technique in a sterilearea. The product is then freeze-dried and the containers are sealedaseptically.

In the respiratory and gastrointestinal systems, CnT binds to mucosalepithelial cells and is actively transcytosed across the mucosa.Compositions suitable for administration to the respiratory tractinclude aerosols, nebulisable solutions or micro-fine powders forinsufflation. In the latter case, particle size of less than 50 microns,especially less than 10 microns, is preferred.

Compositions suitable for topical mucosal application include normalsaline solutions as described above for injections. Other suitablecompositions include gels and creams.

The skin presents a formidable barrier to the application of CnT. CnTmay be mechanically propelled across the dermal barrier with air orwater pressure injectors or in association with micro-pellets. Othersuitable forms of transdermal delivery include iontophoresis. CnT may beencapsulated into liposomes or niosomes to form suitable trans-dermalcompositions.

The method of administration may take many forms, including topical,intra-dermal, sub-cutaneous, trans-cutaneous, intra cavital and byinhalation of the CnT in a suitable carrier. Examples of suchadministration include, but are not limited to contact with absorbentpledgets having CnT absorbed thereon, contact with biodegradablemicropellets having CnT embedded therein. Also included is injection,for example, injection to the nasal mucosa or injection into thepterygoplatine space through the palate, or injection into affectedareas of the surface skin. More invasively, by myringotomy and injectioninto the middle ear space across the tympanic membranes. Alternativelyless invasive methods include by drops into the inner eyelid andinhalation of an aqueous mist containing same.

The physiological reaction reactions dealt with herein include but arenot limited to conditions such as allergic rhinitis, Infectiousrhinitis, vasomotor rhinitis, serous otitis media, sinusitis, asthma,food allergies and allergic dermatitis.

The amount of CnT administered per administration may be, but is notlimited to between about 0.1 and about 1000 units, suitably betweenabout 1 and about 100 units per administration, preferably between about1 and about 20 units.

The invention is also directed to use of a neurotoxin (CnT) derived fromClostridia sp. for the production of a medicament for blocking orreducing physiological reaction in a mammal to the interaction of IgEantibodies present in said mammal upon contact with the correspondingantigen. The formulations of CnT, including BoNT and TeNT, which aresuitable for the purposes designated herein are well known, but have notpreviously been designated for this purpose.

There is a great need for an effective treatment for allergic disorders.It has long been thought that the allergic reaction involved onlyhistamine release by mast cells. Therefore first line therapy forallergy was antihistamines, or more recently the non-sedatingantihistamines. Other therapies are directed to block the effects of themast cell secretions with adrenergic agonists. It is not obvious tothose skilled in the art that a central role in allergic disordersinvolves the autonomic nervous system and that this nerve activity canbe blocked by CnT for a beneficial effect.

It is an object of the invention to provide a treatment for allergicconditions. Included particularly are allergy related rhinitis, asthma,gastroenterititis, serous otitis, sinusitis and dermatitis, and theirrelated conditions (such as sinusitis and serous otitis media that occursecondary to allergy induced mucosal swelling). Treatment is by localapplication of therapeutically effective amounts of CnTs to the bodystructure and/or the nerves and nerve ganglia supplying thesestructures. CnT interfere with the allergic process by:

-   -   1) Directly blocking neuroimmune secretions by the mast cell or        other immune cell types including but not limited to        eosinophils.    -   2) Block the release of neurohumors by mast cells induced by        autonomic nerve activity.    -   3) Decrease neurohumoral release during axonal reflexes.    -   4) Decrease the parasympathetic effector arm of reflex allergic        responses    -   5) Decrease the increased tonic activity of the autonomic        systemic that is related to prior allergic reactions.    -   6) Decrease the enlargement and hypersensitivity of sensory        nerves induced by nerve growth factor and other neurohumors        released during allergic reactions.    -   7) Reverse certain complications of allergic reactions such as        mucosal thickening by decreasing autonomic nerve activity.

The beneficial effect of this treatment in the:

1) Nasal area, is to decrease the symptoms of sneezing, itching, nasalcongestion and post nasal drip, as well as related conditions includingnasal polyps and mucosal thickening, and complications such as sinusitisand serous otitis.

2) Lungs, is to decrease coughing and wheezing (bronchoconstriction,edema) and the non-specific sensory hypersensitivity related to chronicallergic reactions.

3) Eye, is to decrease itching, tearing and redness.

4) Skin, is to decrease itching and swelling.

5) Gastrointestinal tract, is to decrease gastrointestinal dysmotility.

EXAMPLES Example 1 Seasonal Allergic Rhinitis

1a) A 30-year-old male has seasonal allergic rhinitis. In May, prior topollen formation, 30 units of BoNT are topically applied in eachnostril. Specifically, the nasal cavity is sprayed with a solution of 1%lidocaine and ½% neosynephrine to anesthetize and decongest the nasalmucosa, respectively. Then 1 cc of normal saline containing 30 units ofBoNT is sprayed into each nasal cavity. This method of delivery,although commonly used for application of nasal medication, isinherently inefficient as a significant percentage of a sprayedmedication will exit the posterior nasal cavity and be exhaled orswallowed.

1b) In another embodiment, the patient is treated by placing 1×3 cm²cotton pledgets impregnated with 20 units of BoNT onto the medialsurface of the turbinates and left for one hour, then removed by thephysician. This delivery method is more efficient then a spray, howevera significant percentage of the BoNT will not diffuse from the pledgetsto the mucosa.

1c) Alternatively, the patient has topical anesthesia applied only to alocalized area, preferably the anterior end of the inferior turbinate.Then, patient has a 1 cm² cotton pledget impregnated with 10 units ofBoNT placed onto the mucosa of the anterior turbinate for one hour andthen removed. Within the nasal cavity a mucociliary blanket transportsfluids and particles posteriorly, thereby distributing the BoNT. This isan example of applying a BoNT to a localized region of the nasal cavityand using the normal physiology of the nasal cavity for distribution.The routes of mucociliary clearance of the nose and sinus cavities areknown so that other variations of this method are apparent to thoseskilled in the art.

1d) In another embodiment, using a topical biodegradable depot todeliver BoNT over an extended time period to nasal mucosa is used fortreating the patient. Various biodegradable compounds are known in theart that vary in consistency and rate of dissolution.

An example is oxidized cellulose (marketed as Surgicel® by Johnson &Johnson, New Brunswick, N.J.). The oxidized cellulose can bemanufactured with BoNT as an integral component, or the BoNT can beadded to the oxidized cellulose before its clinical use. Surgicel isavailable in the form of a thin flexible sheet that is often cut to fitthe area of the body to which it will be applied. For intranasal use thesize may vary from a few square millimeters to a 4 by 8 cm² sheet thatcould contact the entire exposed mucosa of the nasal cavity. BoNT can beadded to the Surgicel as a lyophilized powder or after reconstitutioninto solution. If added as a powder it can homogeneously applied ontoone side of the Surgicel and the material can be folded. The material isthen moistened with normal saline to bind the material together andimmobilize the BoNT prior to clinical use. In contrast the BoNT canfirst be constituted into solution and then absorbed into the material.

As an example a 2×6 cm² piece of oxidized cellulose is saturated with 1cc of normal saline containing a total of 10 units of BoNT. Using anasal speculum the nostril is dilated and the nasal cavity is visualizedand the saturated cellulose place therein. The cellulose will graduallydissolve over hours while releasing a small continual dose of BoNTdirectly onto the nasal mucosa.

Alternatively 0.5 cc. of normal saline containing 5 units of BoNT isapplied to the oxidized cellulose which is grasped with bayonet forcepsand placed flat onto the medial surface of the anterior end of theinferior turbinate of each nasal cavity. As disclosed above themucociliary action of the nasal mucosa transports liquids and particlesfrom the anterior to posterior nasal cavity.

1e) In another embodiment the patient is injected with BoNT solutiondirectly into the nasal mucosa. After anesthetizing and decongesting themucosa, 5 units of BoNT in 1 cc of saline are injected beneath themucosa throughout the length of the inferior turbinate with a 25 gaugespinal needle coupled to a 1 cc syringe.

1f) Alternatively the patient can be treated with TeNT.

Compositions of TeNT for intranasal administration, can range in dosefrom 0.1 to 1000 units in 0.1 cc to 10 cc of solution. One preferablecomposition is 10 units of TeNT in 1 cc of normal saline.

As an example the same patient is treated by spraying each nostril with1 cc of normal saline solution containing 10 units of TeNT.

Alternatively, if only decongestion is desired 10 units of TeNT can betopically applied.

In another embodiment, 1 unit of recombinant DNA coding for TeNT ispressure injected across the nasal mucosa to transfect mucosal cells.These cells then express the TeNT for months.

Example 2 Perennial Allergic Rhinitis

2a) Direct Injection of the Sphenopalatine Ganglia.

A 40-year-old female has chronic nasal congestion due to perennialrhinitis. Skin testing demonstrates that she is allergic to numerousenvironmental antigens and intranasal examination shows enlargedturbinate mucosa and nasal polyps. Testing of nasal secretions showseosinophilia. An injection of 20 units of BoNT in a 1 cc solution ofnormal saline is made through the sphenopalatine canal into the area ofthe sphenopalatine ganglia.

In another embodiment, 100 units of BoNT embedded in 0.1 micronbiodegradable pellets suspended in solution is injected into the mucosaof each turbinate to slowly release its contents over 6 months.

Alternatively a biodegradable carrier containing 5 units of BoNT isplaced intranasally in the most superior and posterior aspect of thelateral nasal wall. This permits the BoNT to diffuse across the nasalwall to the underlying sphenopalatine ganglia.

Example 3 Serous Otitis Media

A 3-year male has a history of serous otitis media. Under generalanesthesia a myringotomy is made into the tympanic membranes and 2 unitsof BoNT in 0.5 cc of normal saline is injected.

Example 4 Sinusitis

A fifty-year-old male has a history of allergic rhinitis and recurrentsinusitis. Ten units of BoNT are injected through the palate into thepterygopalatine space to block the sphenopalatine ganglion.

Example 5 Allergic Dermatitis

A seventy-year-old male has severe allergic dermatitis of the forearmskin. Each forearm is injected with 10 injections of 0.3 units BoNT in0.1 cc of normal saline. Each injection is made intradermally at 3 cmintervals.

Example 6 Allergic Asthma

A 13-year-old boy has severe allergic asthma. He is treated by bimonthlyinhalation therapy of an aerosilized solution of 5 units of botulinumtoxin in 5 cc of normal saline. Alternatively the same botulinumsolution can be injected directly into the trachea and bronchi. Afterinjecting local anesthesia into the skin overlying the cricothyroidmembrane a needle is passed directly through the skin and cricothyroidmembrane and the BoNT solution is sprayed into the trachea. The solutiondrips down to reach the bronchial mucosa where it is topically absorbed.

Example 7 Allergic Rhinitis

A 40 year old male has allergic rhinitis and an associated reflexchronic cough. Application of CnT to the nasal cavity as described aboveor alternatively injected or aerosilized topical application of BoNT tothe lungs is used to treat the cough.

Example 8 Food Allergies

A 10-year-old boy with food allergies manifested by bloating anddiarrhea is treated with a rectal suppository containing 50 units ofBoNT. The suppository is composed of biocompatible material designed tobe solid at room temperature but to dissolve at body temperature.Sufficient materials for the composition are cocoa butter, glycerinatedgelatin, hydrogenated vegetable oils, polyethylene glycols of variousmolecular weights and fatty esters of polyethylene glycol.

Example 9 Infectious Rhinitis

Chronic allergic exposure causes the nervous system to become hyperresponsive to non-allergic stimulation such as that caused by viralinfections.

A patient with rhinitis is given 10 units of BoNT and 5 units of TeNTprior to spring.

1. A method of blocking or reducing allergic rhinitis in a mammalresulting from the interaction of IgE antibodies present in said mammalupon contact with the corresponding antigen, by the administration tosaid mammal of a therapeutically effective amount of a neurotoxin (CnT)to treat allergic rhinitis, wherein the CnT is isolated or purified froma species of Clostridia selected from the group consisting of C.botulinum, C. butyricum and C. beratti.
 2. The method of claim 1 whereinthe mammal is a member of H. sapiens.
 3. The method of claim 2 whereinthe neurotoxin is isolated or purified from C. botulinum.
 4. The methodof claim 3 wherein the neurotoxins (BoNT) are serotypes A, B, C1, D, E,F or G.
 5. The method of claim 1 wherein CnT is administered by contactwith absorbent pledgets having CnT absorbed thereon.
 6. The method ofclaim 1 wherein CnT is administered by contact with biodegradablecarrier containing CnT.
 7. The method of claim 1 wherein CnT isadministered by injection.
 8. The method of claim 1 wherein CnT isadministered by myringotomy through tympanic membranes.
 9. The method ofclaim 1 wherein CnT is administered by injection into the pterygoplatinespace through the palate.
 10. The method of claim 6 wherein CnT isadministered to pass through the nasal wall to the sphenopalatineganglia.
 11. The method of claim 1 wherein CnT is administered byinhalation of an aqueous mist containing said CnT.
 12. The method ofclaim 1 wherein CnT is administered by injection to the nasal mucosa.13. The method of claim 1 wherein CnT is administered by application ofa suppository containing said CnT.
 14. The method of claim 1 wherein theamount of CnT administered per administration is between about 0.1 andabout 1000 units per administration.
 15. The method of claim 1 whereinthe amount of CnT administered per administration is between about 1 andabout 100 units per administration.
 16. The method of claim 1 whereinthe amount of CnT administered per administration is between about 1 andabout 20 units per administration.
 17. A method of blocking or reducingallergic dermatitis in a mammal resulting from the interaction of IgEantibodies present in said mammal upon contact with the correspondingantigen, by the administration to said mammal of a therapeuticallyeffective amount of a neurotoxin (CnT) to treat allergic dermatitis,wherein the CnT is isolated or purified from a species of Clostridiaselected from the group consisting of C. botulinum, C. butyricum and C.beratti.
 18. A method of blocking or reducing allergic rhinitis in amammal resulting from the interaction of IgE antibodies present in saidmammal upon contact with the corresponding antigen, by the nasaladministration to said mammal of a therapeutically effective amount of aneurotoxin (CnT) to treat allergic rhinitis, wherein the CnT is isolatedor purified from a species of Clostridia selected from the groupconsisting of C. botulinum, C. butyricum and C. beratti and wherein theadministration is selected from the group consisting of: (i) contactwith absorbent pledgets having CnT absorbed thereon; (ii) contact withbiodegradable carrier containing CnT; (iii) myringotomy of CnT throughtympanic membranes; (iv) injection of CnT into the pterygoplatine spacethrough the palate; (v) application of a suppository containing CnT;(vi) an amount of CnT between about 0.1 and about 1000 units peradministration; (vii) an amount of CnT between about 1 and about 100units per administration; and (viii) an amount of CnT between about 1and about 20 units per administration.
 19. The method of claim 1,wherein the administration is topical to the nasal cavity.
 20. Themethod of claim 17, wherein the CnT is incorporated in liposomes. 21.The method of claim 17, wherein the CnT is incorporated in a lyophilizedpowder, liquid solution, cream, ointment, aerosol or liposomes.
 22. Themethod of claim 17, wherein the CnT is administered between about 0.1and about 1000 units per administration.